Darier's Disease (DD), caused by mutations in the endoplasmic reticulum (ER) ATPase ATP2A2 (protein SERCA2b), is characterized by impaired epidermal cell-to-cell adhesion, pathologic apoptosis, and defective keratinocyte differentiation. Current treatment modalities do not address the underlying defects, and are ineffective in many patients. DD also serves as a model to examine Ca2+ control of adherens junctions as well as desmosome formation and turnover, knowledge that is directly applicable to processes such as wound healing or barrier repair. During the previous grant period we discovered that keratinocyte lipid, PKCa and Ca2+ signaling pathways intersect in normal and DD keratinocytes to control Ca2+-dependent differentiation and formation of adherens or desmosomal junctions. Our most recent experiments demonstrate that upregulating glucosyceramide synthase (GCS) or downregulating Sphingosine-1-Phosphate Lyase (SGPL1) or ceramidase rescue DD junctional formation. These findings suggest that the lipid defect in DD centers on synthesis of specific ceramide species such as C16, or subsequent metabolism to downstream metabolites, including sphingosine/S-1-P, Ceramide-1-Phosphate (C1P) or glucosylceramide. We thus have identified three enzyme targets (SGPL1, ceramidase, and GCS) that can be modulated to improve DD, and developed new models to study the suprabasal differentiation, acantholysis and apoptosis characteristic of the DD epidermis. These tools will be used to test our hypothesis: Overall Hypothesis: Keratinocyte ER Ca2+ signaling controls keratinocyte lipid metabolism, which in turn feeds back to enhance ER Ca2+ sequestration and capacitive Ca2+ entry. SERCA2b mutations in DD reduce ER Ca2+ sequestration and impair ceramide and sphingolipid metabolism, hindering PKCa -mediated desmosome formation and E-cadherin-mediated adherens junction formation/capacitive Ca2+ influx. Normalizing lipid metabolism will increase ER Ca2+ levels and will ameliorate DD acantholysis, impaired differentiation and apoptosis. We now propose to: 1) identify and correct the lipid signaling defects induced by defective ER Ca2+ sequestration (Sp. Aim #1); and 2) define how PKCa, intracellular ceramide/sphingolipid and ER Ca2+ signaling pathways interact in normal vs. DD keratinocyte signaling (Sp. Aim 2). The short-term goal of these studies is to develop more effective treatments for DD. The long term goal of these studies is to understand how ER Ca2+ sequestration and release interacts with lipid and PKCa signaling to control keratinocyte and epidermal differentiation, cell-to-cell adhesion and apoptosis.